【摘要】 为阐明GmWRKY50转录因子在大豆与大豆花叶病毒(SMV)互作过程中的作用机制,以大豆叶片的RNA为模板,利用PCR技术克隆了GmWRKY50基因的编码区全长,利用MEGA 7.0软件对不同物种中GmWRKY50的相似氨基酸序列进行比对并进行系统进化树分析,通过原核表达技术,构建pColdⅡ-GmWRKY50重组质粒,转化大肠杆菌BL21后,利用IPTG对带有His标签的GmWRKY50重组蛋白进行诱导,经Ni-NTA柱对重组蛋白进行纯化,收集纯化后的重组蛋白进行多克隆抗体的制备,通过Western Blotting检测GmWRKY50在SMV侵染前后的表达水平变化。结果表明,GmWRKY50基因的CDS全长为495 bp,编码165个氨基酸,在N端含有一个WRKY结构域,属于WRKY DNA-binding结构域蛋白。序列比对及系统进化树分析表明,大豆GmWRKY50与拟南芥AtWRKY50同源关系最近;IPTG浓度为0.5 mmol/L,诱导时间为4 h,重组蛋白的诱导效果良好,利用镍离子亲和纯化获得与His蛋白标签融合的目的蛋白,具有较高的可溶性。以融合蛋白为抗原制备的多克隆抗体可以特异性结合GmWRKY50蛋白,GmWRKY50蛋白在不亲和组合中受SMV侵染诱导而上调表达,为进一步研究转录因子GmWRKY50在大豆抵御SMV侵染过程中的功能奠定了基础。更多还原

【Abstract】 To elucidate the mechanism of the transcription factor GmWRKY50 in the interaction between soybean and Soybean mosaic virus(SMV),the full-length coding region of GmWRKY50 gene was cloned by PCR using RNA of soybean leaves as template, the similar amino acid sequences of GmWRKY50 in different species were compared by MEGA 7.0 and phylogenetic tree analysis was carried out.The recombinant plasmid pColdⅡ-GmWRKY50 was constructed through prokaryotic expression technology.After transformation of Escherichia coli BL21,the GmWRKY50 recombinant protein with His tag was induced by IPTG.The recombinant protein was purified by Ni-NTA column, and the purified recombinant protein was collected for polyclonal antibody preparation.Western Blotting was used to detect the expression level changes of GmWRKY50 before and after Soybean mosaic virus infection.The results showed that the CDS of GmWRKY50 gene was 495 bp in length, encoding 165 amino acids, and contained a WRKY domain at the N terminal, belonging to WRKY DNA-binding domain protein.Sequence alignment and phylogenetic tree analysis showed that soybean GmWRKY50 had the closest homologous relationship with Arabidopsis AtWRKY50.When IPTG concentration was 0.5 mmol/L and induction time was 4 h, the induced effect of recombinant protein was good, and the target protein fused with His protein tag was obtained by nickel ion affinity purification, which has high solubility.The polyclonal antibody prepared with fusion protein as antigen could specifically bind GmWRKY50,GmWRKY50 was up-regulated by SMV infection in incompatible combinations.This study lays the foundation for further investigation of the function of transcription factor GmWRKY50 in soybean resisting SMV infection.更多还原